Effect of dibasol and ascorbic acid on the antiviral activity of human interferon in cell culture]

The antiviral effect of human lymphocytic interferon was studied in the primary culture of human embryonic fibroblasts in the presence of dibazol and ascorbic acid. It was found that dibazol and ascorbic acid in concentrations of 5 and 10 gamma/ml respectively were capable of increasing the antiviral effect of human interferon in homological cells. The assays of 13 lots of interferon showed that its average titer in the experiments with ascorbic acid was 2.5 times higher than that in the control. The assays of 21 lots of interferon showed that its average titer in the experiments with dibazol was 3 times higher than that in the control. It is suggested that an increase in the protective properties of interferon in the presence of dibazol and ascorbic acid is connected with their capacity for stimulating the intracellular production of DNA and protein. The data obtained indicate that dibazol and ascorbic acid may be recommended in the complex of therapy and prophylaxis of antiviral infections.     

Production of cloned human leukocyte interferon by Bacillus subtilis: optimal production is connected with restrained growth

Bacillus subtilis, transformed with a plasmid containing the human alpha-2 (leukocyte) interferon gene, was cultivated in batch and continuous culture in a complex medium. In continuous culture with dissolved oxygen of less than 10% of air saturation, the extracellular interferon titer decreased sharply when the growth rate was lower or higher than the optimal one (mu = 0.14 h-1). Thus, a relatively low growth rate was best for extracellular interferon production, and oxygen limitation enhanced interferon production. The mean output rate in batch culture after successful harvest was 20 X 10(6) IU/liter per h and the maximal output rate in continuous culture was 14 X 10(6) IU/liter per h.     

Gamma interferon enhances the killing of Staphylococcus aureus by human neutrophils

The effect of purified human interferon-gamma on the responsiveness of human neutrophils was investigated. Pre-incubation of neutrophils with 100 U interferon ml-1 for 10 min at 37 degrees C resulted in a 2.5-fold increase in N-formylmethionyl-leucyl-phenylalanine-stimulated reactive oxygen metabolite generation (as assayed by luminol-dependent chemiluminescence). Pre-treatment of neutrophils with interferon also potentiated their ability to kill Staphylococcus aureus, and thus it is proposed that this lymphokine may also enhance neutrophil function in vivo under certain pathological conditions.     

Production of cloned human leukocyte interferon by Bacillus subtilis: optimal production is connected with restrained growth.

Bacillus subtilis, transformed with a plasmid containing the human alpha-2 (leukocyte) interferon gene, was cultivated in batch and continuous culture in a complex medium. In continuous culture with dissolved oxygen of less than 10% of air saturation, the extracellular interferon titer decreased sharply when the growth rate was lower or higher than the optimal one (mu = 0.14 h-1). Thus, a relatively low growth rate was best for extracellular interferon production, and oxygen limitation enhanced interferon production. The mean output rate in batch culture after successful harvest was 20 X 10(6) IU/liter per h and the maximal output rate in continuous culture was 14 X 10(6) IU/liter per h.     

Analysis of the herpes simplex virus genome during in vitro latency in human diploid fibroblasts and rat sensory neurons.

We have previously designed in vitro model systems to characterize the herpes simplex virus type 1 (HSV-1) genome during in vitro virus latency. Latency was established by treatment of infected human embryo lung fibroblast (HEL-F) cells or rat fetal neurons with (E)-5-(2-bromovinyl)-2'-deoxyuridine and human leukocyte interferon and was maintained by increasing the incubation temperature after inhibitor removal. Virus was reactivated by reducing the incubation temperature. We have now examined the HSV-1-specific DNA content of latently infected HEL-F cells and rat fetal neurons treated with (E)-5-(2-bromovinyl)-2'-deoxyuridine and human leukocyte interferon and increased temperature. The HEL-F cell population contained, on an average, between 0.25 and 0.5 copies of most, if not all, HSV-1 HindIII and XbaI DNA fragments per haploid cell genome equivalent. In contrast, the latently infected neurons contained, on an average, 8 to 10 copies per haploid cell genome equivalent of most HSV-1 BamHI DNA fragments. There was no detectable alteration in size or molarity of the HSV-1 terminal or junction DNA fragments obtained by HindIII, XbaI, or BamHI digestion of the latently infected neuron or HEL-F cell DNA, as compared with digestion of a reconstruction mixture of purified HSV-1 virion and HEL-F cell DNAs. These data suggest that the predominant form of the HSV-1 genome in either latently infected cell population is nonintegrated, linear, and nonconcatameric.     

Interferon effects on microfilament organization cellular fibronectin distribution, and cell motility in human fibroblasts

We have shown previously (Pfeffer et al., 1979, Exp. Cell Res. 121:111-120) that treatment of human fibroblasts, planted at a density of 2x10(3) cells/cm(2), with purified human fibroblasts interferon (640 U/ml) for 3 d at 37 degrees C decreases the overall rate of cell proliferation to 35-40 percent of the control value. In the present experiments we have characterized the phenotype of interferon-inhibited fibroblasts. The mean volume of trypsinized, interferon-treated cells was increased 31 percent abover that of control cells. The interferon-treated population was much more heterogeneous than the control population with respect to volume, and there was a considerable overlap in the volume distributions of the two populations. The cell surface area was, on the average, increased 65 percent after interferon treatment. More than 80 percent of the treated cells had enlarged nuclei, many of which were lobed, and the fraction of binucleated cells was increased fivefold. After interferon treatment, over 40 percent of the cells showed large actin-containing fibers in the form of multiple parallel arrays. Fewer than 5 percent of the control cells contained such large actin fibers. The number of actin fibers of all sizes was tripled in the treated fibroblasts on a per cell basis and, calculated per unit surface area of the cells, the number was increased 82 percent. In contrast, 10-nm filaments and microtubules did not appear to be increased in number per unit surface area of the cells. The increases per cell in the abundance of these structures were directly related to increased cell size. After interferon treatment, fibronection was distributed in arrays of long filaments covering most portions of the cell surface. Interferon treatment markedly decreased the rate of cell locomotion as well as membrane ruffling and saltatory movements of intracellular granules.     

Effect of interferon on the yield of chromosome aberrations in cultured human lymphocytes irradiated with fast neutrons

A study was made of the influence of leucocytic interferon on the yield of chromosome aberrations in a human lymphocyte culture after irradiation of cells with fast neutrons (0.85 MeV) at the G0- and G1-stages. It was shown that in cells treated with interferon (50 UE/ml) prior to irradiation the total yield of aberrations and of some of their types was invariable as compared to the irradiated control.     

Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor confers resistance to the antiproliferative effect of interferon-alpha

BACKGROUND: Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a 442 amino acid polypeptide-designated viral interferon regulatory factor (vIRF) that displays homology to members of the interferon regulatory factor (IRF) family that bind to consensus interferon sequences and transactivate cellular genes that can modulate growth inhibition. Studies were conducted to determine whether vIRF affects the growth suppression mediated by interferon-alpha (IFN-alpha) in a human B lymphocyte cell line. MATERIALS AND METHODS: The human B lymphocyte cell line Daudi, which is sensitive to the antiproliferative effects of IFN-alpha, was stably transfected to express vIRF, and the proliferative response of vIRF expressing cells to IFN-alpha was compared with controls. The effect of vIRF on IRF- 1 transactivation was analyzed by co-transfection of an IFN-alpha-responsive chloramphenicol acetyltransferase reporter and expression plasmids encoding IRF-1 and vIRF. Electrophoretic mobility shift assays were conducted to determine whether vIRF interferes with the DNA binding activity of IRF-1. RESULTS: Daudi human B lymphocyte cells expressing vIRF were resistant to the antiproliferative effects of IFN-alpha, whereas wild-type Daudi or Daudi cells transformed with vector DNA were growth inhibited by IFN-alpha. The activation of an interferon-responsive reporter by IFN-alpha or IRF-1 was repressed by expression of vIRF. IRF-1 DNA binding activity was unaffected by vIRF, and vIRF alone did not bind to the interferon consensus sequence. CONCLUSIONS: These studies revealed that vIRF functions to inhibit interferon-mediated growth control of a human B lymphocyte cell line by targeting IRF-1 transactivation of interferon-inducible genes. Since KSHV is a B lymphotropic herpesvirus associated with two forms of B lymphocyte neoplasms, these effects of vIRF likely contribute to B cell oncogenesis associated with KSHV infection.     

玉屏风颗粒联合重组人干扰素α-1b雾化吸入治疗对反复呼吸道感染患儿炎性因子和T细胞亚群的影响

目的:探讨在重组人干扰素α-1b雾化吸入治疗的基础上加用玉屏风颗粒对反复呼吸道感染(RRTI)患儿炎性因子和T细胞亚群的影响。方法:将我院于2018年3月~2020年2月期间收治的RRTI患儿180例根据信封抽签法分为对照组(n=90)和实验组(n=90),均给予止咳、退热、平喘、抗感染等常规治疗的基础上,对照组患儿予以重组人干扰素α-1b雾化吸入治疗,实验组患儿则在对照组的基础上联合玉屏风颗粒治疗,比较两组患儿疗效、症状消失时间、炎性因子、T细胞亚群和不良反应。结果:实验组治疗3周后的临床总有效率为91.11%(82/90)高于对照组的78.89%(71/90)(P<0.05)。实验组肺部啰音、发热、喘息、咳嗽症状消失时间较对照组短(P<0.05)。两组治疗3周后血清白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、白介素-10(IL-10)水平均下降,且实验组低于对照组(P<0.05)。两组治疗3周后CD8⁺较治疗前降低,且实验组低于对照组(P<0.05);CD4⁺/CD8⁺、CD3⁺、CD4⁺均较治疗前升高,且实验组高于对照组(P<0.05)。比较两组不良反应发生率未见统计学差异(P>0.05)。结论:玉屏风颗粒联合重组人干扰素α-1b雾化吸入治疗RRTI患儿,可迅速改善患儿临床症状、T细胞亚群以及炎性因子水平,且不增加不良反应发生率,疗效令人满意。     

Virus replication and high-titered interferon production in human leukocyte cultures inoculated with Newcastle disease virus

Wheelock, Frederick E. (Western Reserve University, Cleveland, Ohio). Virus replication and high-titered interferon production in human leukocyte cultures inoculated with Newcastle disease virus. J. Bacteriol. 92:1415-1421. 1966.-High titers of interferon (20,480 culture-protecting units per ml) are produced in freshly prepared human leukocyte cultures inoculated with a Newcastle disease virus (NDV)-cell multiplicity of 1:1. NDV replicates to low titers in these cultures. Incubation of leukocytes at 37 C for 24 hr prior to inoculation of NDV results in almost complete loss of detectable interferon production, but virus replicates to higher titers than in the freshly prepared cultures. In contrast, no diminution of interferon production in response to phytohemagglutinin (PHA) occurs on 24 hr of incubation of cultures prior to addition of PHA. Experiments with cultures of predominantly pure cell fractions of peripheral blood indicate that the lymphocyte fraction produces interferon in response to either NDV or PHA, and that polymorphonuclear leukocytes produce no interferon in response to these agents. These studies suggest a hitherto unsuspected ability of human lymphocytes to produce high titers of interferon in vivo.     

Regulation by interferon beta-1a of reactive oxygen metabolites production by lymphocytes and monocytes and serum sulfhydryls in relapsing multiple sclerosis patients

The activation of lymphocytes and monocytes and the concentration of reduction equivalents in serum were studied in a cohort of multiple sclerosis (MS) patients undergoing weekly treatment with 30 microg intramuscular interferon beta-1a for 2 years. The degree of activation of monocytes and lymphocytes and reactive oxygen species (ROS) production was higher in MS patients than in healthy controls and decreased in the course of interferon beta-1a treatment approaching control values. The concentration of reduced sulfhydryls in the serum of MS patients was lower than in healthy controls and the treatment with interferon beta-1a (IFNbeta-1a) raised the levels approaching the values of healthy controls.     

Induction of human HL-60 leukemic cell differentiation by immune interferon is accompanied by an increase in NADase activity and by a decrease in DNA-binding proteins

The effects of highly purified human immune interferon (IFN-gamma) on the differentiation of human promyelocytic HL-60 leukemic cells have been studied. The addition of 100 units/ml interferon to HL-60 cells for 5 days results in morphological changes characteristic of macrophages. At the biochemical level, there is a 3-fold increase in the specific activity of the enzyme NADase. Kinetic analysis shows that IFN-gamma causes an increase in the Vmax of NADase without affecting the apparent Km. Pulse labeling experiments with [35S] methionine show a marked change in the de novo synthesis of several proteins in the course of interferon treatment. Chromatography on DNA-agarose show that after treatment with interferon for 24 or 48 h, there is a 60-70% decrease in newly synthesized proteins which bind DNA-agarose and can be subsequently displaced from the column with 2% SDS containing buffer (from 7.7-8.7% bound in control cell extracts to 2.6-3.1% bound in interferon treated cell extract).     

Effect of human interferons on cAMP phosphodiesterase activity in a culture of human ovarian carcinoma cells (CaOv)

Crude and purified human interferons of alpha type exerted 2 step inhibition of cAMP phosphodiesterase activity in CaOv cells: in 4 and 24 hours after cells treatment with interferon. The maximal inhibition was obtained in response to interferon doses 1200-2000 IU/ml. In contrast to natural interferons the human alpha 2 recombinant interferon (20-25000 IU/ml) did not inhibit the cAMP phosphodiesterase activity in CaOv cells.     

Effect of mycoplasma on interferon production and interferon assay in cell cultures

Armstrong, D. (The Children's Hospital of Philadelphia, Philadelphia, Pa.), and K. Paucker. Effect of mycoplasma on interferon production and interferon assay in cell cultures. J. Bacteriol. 92:97-101. 1966.-The influence of mycoplasma on the production and action of interferon was studied in cultures of both L and human embryonic kidney (HEK) cells. Mycoplasma hominis 1, the Negroni agent, and the F12 mycoplasma were used for infection of L cells, and M. hominis 1 and M. pneumoniae for inoculation of HEK cells. All strains were capable of multiplication in the culture systems employed. None produced detectable levels of interferon, and responsiveness of the cells to induction of interferon by virus remained unaltered. Infection with mycoplasma did not impair the sensitivity of the cells to the action of interferon, nor was the replication of vesicular stomatitis virus noticeably diminished.     

高效液相色谱法测定重组人干扰素α-2b注射液中乙二胺四乙酸二钠含量

目的:利用高效液相色谱(HPLC)法测定重组人干扰素α-2b注射液中EDTA二钠(乙二胺四乙酸二钠)的含量。方法:将EDTA二钠与氯化铁溶液于70℃水浴中反应20 min左右;色谱柱为SunFire C18(250 mm×4.6 mm,5μm,Waters),流动相为5%甲醇+95%0.64 g/L四丁基溴化铵和4.1 g/L三水合乙酸钠混合液,用冰乙酸调pH值至4.0,流速1 mL/min,检测波长254 nm。结果:该测定方法线性范围为0.025~0.5 g/L,线性关系良好(r=0.9997),加样回收率为98.27%(n=9,RSD=2.55%)。结论:本方法准确、快速、可靠,可用于重组人干扰素α-2b注射液中EDTA二钠含量的测定。     

Binding of human interferon alpha to cells of different sensitivities: studies with internally radiolabeled interferon retaining full biological activity.

The characteristics of interferon binding to various cells with different interferon sensitivity were studied by using [3H]leucine-labeled, pure human interferon alpha from Namalwa cells. Scatchard analysis of the binding data on cells sensitive to interferon alpha (human FL and fibroblasts and bovine MDBK) indicated the presence of two kinds of binding sites with high and low affinities. The binding constants of the high-affinity sites in these cells were similar (4 X 10(10) to 11 X 10(10) M-1). Cells insensitive to human interferon alpha (human HEC-1 and mouse L cells) were shown to have only low-affinity sites, suggesting that high-affinity binding sites are indispensable for interferon sensitivity and represent interferon receptors. However, the number of sites in three human diploid fibroblast strains and one strain trisomic for chromosome 21 were not proportionally correlated to the interferon sensitivity of the cells. The high-affinity binding to human cells was completely inhibited by both nonradioactive human interferons alpha and beta in a similar manner, but binding to bovine MDBK cells, on which human interferon beta is practically inactive, was inhibited effectively only by interferon alpha and not by beta. These results suggest that the receptor for human interferon alpha is common to human interferon beta in human cells, whereas the receptor on bovine cells binds only human interferon alpha.     

Regulation of Human Immunodeficiency Virus Replication by 2′,5′-Oligoadenylate-Dependent RNase L

Activation of RNase L by 2′,5′-linked oligoadenylates (2-5A) is one of the antiviral pathways of interferon action. To determine the involvement of the 2-5A system in the control of human immunodeficiency virus type 1 (HIV-1) replication, a segment of the HIV-1 nef gene was replaced with human RNase L cDNA. HIV-1 provirus containing sense orientation RNase L cDNA caused increased expression of RNase L and 500- to 1,000-fold inhibition of virus replication in Jurkat cells for a period of about 2 weeks. Subsequently, a partial deletion of the RNase L cDNA which coincided with increases in virus production occurred. The anti-HIV activity of RNase L correlated with decreases in HIV-1 RNA and with an acceleration in cell death accompanied by DNA fragmentation. Replication of HIV-1 encoding RNase L was also transiently suppressed in peripheral blood lymphocytes (PBL). In contrast, recombinant HIV containing reverse orientation RNase L cDNA caused decreased levels of RNase L, increases in HIV yields, and reductions in the anti-HIV effect of alpha interferon in PBL and in Jurkat cells. To obtain constitutive and continuous expression of RNase L cDNA, Jurkat cells were cotransfected with HIV-1 proviral DNA and with plasmid containing a cytomegalovirus promoter driving expression of RNase L cDNA. The RNase L plasmid suppressed HIV-1 replication by eightfold, while an antisense RNase L construct enhanced virus production by twofold. These findings demonstrate that RNase L can severely impair HIV replication and suggest involvement of the 2-5A system in the anti-HIV effect of alpha interferon.     

地高辛标记探针检测重组人干扰素β_(1b)中DNA残留量

为检测注射用重组人干扰素β1b半成品中外源性DNA残留量,以重组人干扰素β1b工程菌基因组DNA为模板,用地高辛标记探针,并以此探针进行点杂交。结果证明,该方法检测灵敏度较好,特异性较强,操作较安全简便,可用于重组人干扰素β1b制备过程中的质量监控及半成品的检定。     

Interferon levels in human pulmonary tumors are lower than plasma levels

It is uncertain whether interferon levels in the interstitial fluid of tumors are equivalent to interferon plasma levels and we have investigated this problem in human pulmonary tumors by infusing human recombinant interferon alpha A and natural interferon Beta for about three hours before surgery. By determining the hematocrit and hemoglobin content it was possible to calculate interferon values (International Units/g wet tissue) present in the interstitial fluid of tumor and lung samples, simultaneously. In 14 patients (epidermoids, n = 9 and adenocarcinomas, n = 5) interferon levels in tumor and "normal" lung expressed as percentages of interferon plasma levels were: 9.5 +/- 3.9 and 29.8 +/- 6.9 for recombinant interferon alpha A and 3.1 +/- 0.4 and 10.1 +/- 2.4 for natural interferon Beta, respectively. Differences for both interferons are statistically significant (p less than 0.05). To our knowledge these are the first data indicating that interferon levels in pulmonary tumor interstitial fluid are markedly lower than those in normal lung although they do not clarify the main factor responsible for the decrease, they explain at least in part the negligible therapeutic activity of interferons in these tumors and emphasize the need for new approaches for improving the therapeutic index of interferons.